The present invention relates to a method and agents for the immunological determination of enzymes, in particular for the quantitative immunological determination of isoenzymes of acid or alkaline phosphatase in body fluids.
In recent years, quantitative determination of isoenzymes in blood has gained considerable importance for differential diagnosis of various internal illnesses in humans. For example, the concentration of certain isoenzymes of alkaline phosphatase, of acid phosphatase, of lactate dehydrogenase or of galactosyl transferase in the blood of tumor patients is significantly increased. Quantitative determination of these enzymes provides clinical chemistry with important parameters for recognizing tumors early, observing their progress and monitoring the success of the therapy.
It is known that acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) of humans occurs in the form of several isoenzymes. The concentration of the prostate isoenzyme of acid phosphatase in the blood of many patients with prostate tumors is significantly increased. The detection of this isoenzyme is thus of great importance for recognizing early stages of the illness and for its therapy.
Several isoenzymes which can be differentiated biochemically have also been described for alkaline phosphatase (AP) (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) of humans. Quantitative determination of the isoenzymes in the blood which originate from the liver and bones or from the placenta is important in clinical chemistry. The identification and quantitative determination in urine of so-called cytoplasmatic small intestine AP from the kidneys has also recently appeared to be interesting for the diagnosis of kidney diseases. The placenta isoenzyme of alkaline phosphatase occurs in the blood of pregnant women. an enzyme which reacts identically from a biochemical and immunological point of view, the so-called Regan isoenzyme of alkaline phosphatase, has been found in increased concentrations in the blood of patients with various tumors, and in particularly high concentrations in cases of tumors of the testicles or of the ovaries.
Plasma or serum usually contains mixtures of isoenzymes of the phosphatases mentioned. A simple measurement of the total activity of acid or alkaline phosphatase therefore gives no clear information regarding the occurrence and the activity of the isoenzyme components specifically sought in the blood. Furthermore, the isoenzymes of interest frequently represent only a small proportion of the total activity in the blood, so that determination of their activity without prior concentration of the sample is very inaccurate. Yet, aliquots of the biological samples are employed for many determinations of the activity of isoenzymes of alkaline phosphatase or of the prostate isoenzyme of acid phosphatase, without the enzyme first being separated off from the sample liquid. Moreover, the fact that the samples can contain interference factors, such as, for example, enzymes which interfere with the test or coagulation-inhibiting additives or metabolites of pharmaceuticals, is not taken into consideration (Clin. Chem. 21, 1 D-432 D (1975)) and these further affect the test reliability.
The conventional methods for the determination of the prostate isoenzyme of acid phosphatase measure the amount of a chromophor liberated from a chromogenic substrate by the action of the enzyme. In more recent methods, specific antibodies against acid phosphatase are used for recognition and quantitative detection. These immunological methods, such as electroimmunodiffusion, countercurrent immunoelectrophoresis, radioimmunoassay and fluorescence-immunoassay, however, have considerable disadvantages. For example, electroimmunodiffusion is tedious and is unsuitable for routine determinations. Countercurrent immunoelectrophoresis gives only semiquantitative results. Radioimmunoassay is time-consuming and is disadvantageous because of the use of radioactive substances. Carrying out the radioimmunoassay in a special laboratory, the danger of radiation and the low lifetimes of the radioactively labelled reagents are particularly disadvantageous. Fluorescence-immunoassay is relatively complicated from a technical point of view and is susceptible to errors because of the need to pipette a particular reagent; this causes metering problems. In addition, it can be only partly mechanized and special measurement instrumentation is required.
The determination of alkaline phosphatase from the placenta in the serum of pregnant women and of tumor patients (Regan isoenzyme) has hitherto been chiefly carried out by measuring the enzyme activity either after subjecting the sample to heat treatment or in the presence of inhibitors, or by differentiating the isoenzyme distribution by a combination or preliminary heat treatment of the sample and measurement in the presence of inhibitors. These methods have the disadvantage that they are too insensitive and are not sufficiently selective. The results achieved with electrophoretic methods on various carriers are also unsatisfactory because they do not permit a quantitative result. In some cases they yield artifacts and they cannot be used as routine determinations.
In more recent methods, specific antibodies against the isoenzymes of alkaline phosphatases have been employed for recognition and for quantitative detection. Precipitation of the AP isoenzymes by specific antibodies in the free or immobilized form is insensitive and susceptible to interference. In other techniques, the AP is bonded to covalently cross-linked antiserum and measured in the bonded form. The disadvantages of this technique are the expensive preparation of the reagent, its poor meterability and the troublesome separation by centrifugation. Other immunological methods, such as simple radioimmunodiffusion, electroimmunodiffusion and radioimmunoassay have the same disadvantages described above for the determination of acid phosphatase.
A process for the immunological determination of the prostate isoenzyme of acid phosphatase based on a solid phase fluorescence-immunoassay is known, for example, from PCT Application No. WO 79/00475. In this process, specific antibodies against this isoenzyme are first covalently bonded to a Sepharose gel activated with a cyanogen halide. The active groups remaining on the Sepharose gel must then be blocked and the excess blocking reagent must be removed, which requires additional washing steps. The prostate isoenzyme of acid phosphatase which is bonded to the antibodies, is then determined by measuring the fluorescence of a product formed when the substrate is split.
Apart from the disadvantages already described in connection with evaluation via fluorescence measurement, this process also has the considerable disadvantage that the antibodies must first be covalently bonded to a carrier by expensive methods. Using a carrier in particle form, furthermore, introduces the problems of the poor meterability of a particle suspension and of centrifugation steps in carrying out the determination.
Processes are also known in which an immunological component can be bonded to a water-insoluble carrier not only covalently but also by adsorption (German Offenlegungsschrift No. 2,901,391 and U.S. Pat. No. 4,106,043). In these cases, however, the bonding partner consists of a covalent coupling product of the antigen or antibody and an enzyme, so that a covalent bond must also first be formed here.